Vascular stiffness has been clearly established as a risk factor for cardiovascular disease, and is an independent predictor of cardiovascular morbidity and mortality. Aging is associated with increased vascular stiffness and isolated systolic hypertension, which results from alterations in the properties of all elements of the vascular wall including endothelium, vascular smooth muscle, and matrix. Although both dynamic (alterations in endothelial function and effects on vascular smooth contractility), as well as structural (eg. fracturing of elastin) have been described in aging, molecular mechanisms underlying age-related vascular stiffness remain poorly understood. Thus targeted therapy remains elusive. Tissue transglutaminase (TG2, tTG), expressed in vascular endothelial, smooth muscle cells, and fibroblasts, enzymatically form cross-links between extracellular matrix proteins, and may contribute to this pathobiology. Ca2+dependent activation of TG2 is dependent on its externalization to the cell surface. Recently, it has been demonstrated that S- nitrosylation, a redox-sensitive post-translational modification of cysteine residues, leads to TG2 enzyme inhibition. In Preliminary Data, we demonstrate that TG2 is S-nitrosylated in cellular models and that this leads to decreased cell-surface localization and decreased cross-linking activity. Furthermore, using TG2-/- mice, we show that TG2 is the primary TGase mediating stiffness of conduit arteries, and that it is regulated by endothelium-derived NO. We further demonstrate that in aging rat and human aorta, TG2 activity is increased, and it's S-nitrosylation is decreased despite unchanged TG2 abundance. Finally, inhibition of TG in old rats reduces vascular stiffness. It is now well established that NO bioavailability is diminished and reactive oxygen species (ROS) are increased in aging. Given this change in the nitroso-redox balance, we hypothesize that aging is associated with decreased TG2 S-nitrosylation and therefore, increased externalization and increased matrix cross-linking activity and TG2-dependent downstream signaling. Together, these result in increased vessel stiffness, and ultimately impaired vascular function, a hallmark of aging. In this grant, we propose to determine the role of NO in the regulation of TG2 location, activity and downstream signaling, and determine whether this enzyme is a critical target in age-related vascular stiffness. We will use a hierarchical approach including endothelial cells, vascular tissue from young and old Fischer 344 rats and TG2-/- and NOS3-/- mice, and invasive and non-invasive measures of vascular characteristics in these animal models. The following are the specific aims: 1) To determine the role of endothelium-dependent NO in the of regulation of TG2 subcellular distribution and activity. 2) To determine the role of TGF2 in downstream TG2 mediated vascular signaling. 3) To determine the role of TG2 and its regulation by NO in age-related vascular stiffness in animal models using sophisticated measures of vascular properties in vivo. 4) To determine TG2 activity in the aorta of aging humans. PUBLIC HEALTH RELEVANCE: As we age, our blood vessels become stiffer because of the changes that occur in all wall of the large blood vessels. When the aorta stiffens it is less able to buffer the pulse created by pumping of blood into it from the heart leading high blood pressure in the elderly which is very difficult to treat. This vascular stiffening and high blood pressure is associated with an increase risk of heart attack and stroke as we age. We have identified an enzyme that creates "bridges" or "scaffolds" between other proteins in the vessel wall contributing to its stiffening, and plan to study its mechanism of regulation, thereby helping us to determine whether blocking the enzyme might be important in treating age-related high blood pressure.